Levodione reductase

Abstract

A levodione reductase having the following physical properties is provided: molecular weight: from about 142,000 to about 155,000±10,000 (consisting of four homologous subunits having a molecular weight of 36,000±5,000); co-factor: nicotinamide adenine dinucleotide (AND/NADH); substrate specificity: active on levodione; optimum temperature: about 15° C. to about 20° C. at pH 7.0; optimum pH: 7.5; and activator: K+, Cs+, Rb+, Na+ and NH4+. The levodione reductase according to the present invention produces actinol, an important intermediate for the production of zeaxanthin, from levodione. This enzyme may be produced from a microorganism belonging to the genus Corynebacterium, preferably the microorganism Corynebacterium aquaticum AKU 611 (FERM BP-6448) or a functional equivalent, subculture, mutant or variant thereof. Also provided is a process for producing the levodione reductase that includes cultivating a microorganism of the genus Corynebacterium in an aqueous medium under aerobic conditions, disrupting the cells of the microorganism and isolating and purifying the levodione reductase from the cell-free extract. A process for producing actinol from levodione is also provided that includes contacting levodione with levodione reductase in the presence of the reduced form of nicotinamide adenine dinucleotide or a cell-free extract of the microorganism, and then isolating the resulting actinol from the reaction mixture.

We claim:  
     
       1. An isolated and purified enzyme having levodione reductase activity wherein the enzyme comprises the following physico-chemical properties; 
       (a) a molecular weight of about 142,000 to about 155,000±10,000 measured by gel filtration;  
       (b) a nicotinamide adenine dinucleotide (NAD/NADH) co-factor,  
       (c) catalyzes the reduction of levodione to actinol;  
       (d) an optimum temperature of about 15° C. to about 20° C. at a pH of about 7.0;  
       (e) an optimum pH of about 7.5; and  
       (f) wherein the enzyme is activated by a metal ion selected from the group consisting of K + , Cs + , Rb + , Na +  and NH 4   + .  
     
     
       2. The enzyme according to  claim 1  consisting essentially of four homologous subunits each having a molecular weight of about 36,000±5,000 measured by SDS-polyacrylamide gel electrophoresis. 
     
     
       3. The enzyme according to  claim 1  wherein the enzyme is obtained from Corynebacterium. 
     
     
       4. The enzyme according to  claim 3  wherein the Corynebacterium is selected from the group consisting of  Corynebacterium aquaticum  AKU 611 (FERM BP-6448), and mutants thereof. 
     
     
       5. The enzyme according to  claim 4  wherein the Corynebacterium is  Corynebacterium aquaticum  AKU 611 (FERM BP-6448). 
     
     
       6. An isolated and purified levodione reductase obtained from  Corynebacterium aquaticum  AKU 611 (FERM BP-6448) cells having the following properties: 
       (a) a molecular weight of about 142,000 to about 155,000±10,000 measured by gel filtration;  
       (b) a NAD/NADH cofactor;  
       (c) catalyzes the reduction of levodione to actinol;  
       (d) an optimum temperature of about 15° C. to about 20° C. at a pH of about 7.0;  
       (e) an optimum pH of about 7.5; and  
       (f) the levodione reductase being activated by a metal ion selected from the group consisting of K + , Cs + , Rb + , Na + , and NH 4   + .  
     
     
       7. The levodione reductase according to  claim 6  produced by the process comprising: 
       (a) culturing the  Corynebacterium aquaticum  AKU611 (FERM BP-6448) cells in a nutrient medium under aerobic conditions; and  
       (b) preparing a cell free extract containing the levodione reductase by disrupting the cells.  
     
     
       8. The levodione reductase according to  claim 7  wherein the levodione reductase is isolated from the cell free extract.